CN103412129A - Screening method for optimal concentration ratio of two Bt proteins for cotton bollworm prevention and control - Google Patents

Abstract

The present invention relates to a method for screening the optimal concentration ratio of two Bt proteins for preventing and controlling cotton bollworms. The method comprises: (1) selecting two Bt proteins for preventing and controlling cotton bollworms; (2) inoculating cotton bolls with different resistance levels Insect larvae, calculate the optimal concentration range of the mixed use of the two Bt proteins when controlling cotton bollworm larvae with different resistance levels; (3) Set the ratio of different concentration gradients of the two Bt proteins according to the optimal concentration range, and inoculate with different resistance levels (4) Observe the death of larvae, calculate the mortality, LC ₅₀ and co-toxicity coefficient CTC, and determine the control effect of different concentration ratios of the two Bt proteins. The method of the present invention is highly operable, accurate and efficient, and can clarify the concentration ratio of the two Bt proteins for cotton bollworms in different regions and with different resistance levels, and can be used in the ratio of Bt proteins or other proteins in trans-bivalent cotton Widely used in the selection of insecticides to increase insecticidal effect and delay resistance.

Description

The Screening Method of Optimum Concentration Ratio of Two Bt Proteins for Controlling Cotton Bollworm

technical field

The invention relates to the technical field of biological detection, in particular to a method for screening the optimal concentration ratio of two Bt proteins for preventing and controlling cotton bollworms with different resistance levels.

Background technique

Cotton is a strategic material related to the national economy and people's livelihood, and it is also the second largest crop after grain. Cotton bollworm is one of the main pests that harm cotton fields. The outbreak of cotton bollworm in the early 1990s caused huge economic losses to the national economy of our country. In order to effectively prevent and control cotton bollworm, transgenic cotton was commercially planted in the United States in 1996, and its planting area has grown rapidly around the world. Commercial planting began, and in 2011 the proportion of genetically modified cotton in China reached 71.5%.

While the widespread planting of Bt cotton has brought huge benefits, the resistance of pests to Bt has also become a key factor affecting the sustainable development of transgenic cotton. Tabashnik et al. reported in 2008 that the cotton bollworm in the southeastern United States had developed resistance to Bt cotton in the field; in 2010, it was reported that India began to use the first generation of transgenic cotton in 2002, and by 2009 the pink bollworm in the field had developed resistance to Cry1Ac . Therefore, many international breeding companies have successively developed the second generation of transgenic cotton, such as Bollgard II from Monsanto (expressing Cry2Ab and Cry1Ac proteins), WideStrike from Dow AgroSciences (expressing CrylF and CrylAc proteins), and VipCot from Syngenta (expressing Vip3A and Cry1Ab proteins). )wait.

In addition to expanding the insecticidal spectrum, transgenic bivalent insect-resistant cotton can also delay the development of target pest resistance. Therefore, if the two expressed proteins reach the optimal concentration ratio in the transgenic insect-resistant cotton, then such transgenic cotton can not only kill the target pests more effectively, but also have obvious effects on the resistant pests that may be produced. governance role. The preparation process of transgenic plants is a complex and lengthy process, which is affected by many factors. Due to the influence of gene mutation, the effect of transgenic plants is uncertain, and a lot of screening work is required. Therefore, before the new gene is transferred to cotton, the effect of its new protein is evaluated indoors to determine the optimal concentration ratio, which can save a lot of manpower, material resources and financial resources.

The inventor determined "a method for evaluating the effect of cotton bollworm resistance control (patent application number: 201210480447.4)" in the early stage, and can basically determine the optimal concentration range when the two proteins are mixed. Therefore, on this basis, according to the two The optimal concentration range of protein is mixed with different concentration ratios to determine the optimal concentration ratio for different resistant and sensitive cotton bollworms, which is of great significance to actual production.

Contents of the invention

In order to solve the above problems, the object of the present invention is to provide a screening method for the optimal concentration ratio of two Bt proteins for preventing and treating cotton bollworms, and to determine the optimal concentration ratios of the two Bt proteins for cotton bollworms with different resistance levels .

The screening method for the optimal concentration ratio of two Bt proteins for preventing and treating cotton bollworm provided by the invention, said method comprising the following steps:

(1) Select two Bt proteins for the control of cotton bollworm;

(2) Inoculate cotton bollworm larvae with different resistance levels, and calculate the optimal concentration range of the mixed use of the two Bt proteins when controlling cotton bollworm larvae with different resistance levels;

(3) According to the optimal concentration range, different concentration gradient ratios of the two Bt proteins were set, and cotton bollworm larvae with different resistance levels were inoculated for bioassay;

(4) Observe the death of larvae, calculate the mortality rate, LC ₅₀ and co-toxicity coefficient (CTC), and determine the control effect of different concentration ratios of the two Bt proteins.

In step (1), the two Bt proteins are any two of Cry2Ab protein, Cry1Ab protein, Cry1F protein, Vip3A protein and Cry1Ac protein or other novel Bt proteins.

Preferably, the two Bt proteins are Cry2Ab protein and Cry1Ac protein.

In step (2), the method for calculating the optimum concentration range for the mixed use of the two Bt proteins includes the following steps:

a, select two kinds of Bt protein for preventing and treating cotton bollworm;

b. Inoculation of cotton bollworm larvae with different resistance levels;

c, observe the larva death situation, calculate the mortality rate;

d. Using the two-factor D-saturation method to establish a regression equation, evaluate the control effects of the two Bt proteins, and obtain the optimal concentration range for the mixed use of the two Bt proteins.

For the calculation method of the optimal concentration range of the mixed use of the two Bt proteins, refer specifically to the method of the invention patent application with the application publication number CN102981001A (the application number is 201210480447.4, and the invention name is a method for evaluating the resistance control effect of cotton bollworm).

In steps (2) and (3), the cotton bollworm larvae with different resistance levels are the newly hatched larvae of the sensitive strain or the newly hatched larvae of the resistant strain. Preferably, the sensitive strain of cotton bollworm is 96S; the resistant strain of cotton bollworm is BtR ₁ strain Cry1Ac resistant cotton bollworm. The Cry1Ac-resistant cotton bollworm of the BtR ₁ strain is screened by adding gradually increasing doses of Cry1Ac protein to the artificial diet of cotton bollworm. It has been screened for 125 generations, and its resistance to Cry1Ac is 2199.83 times that of sensitive strains.

In step (3), within the optimal concentration range, set the ratio of different concentration gradients of the two Bt proteins for bioassay; preferably, within the optimal concentration range, set the ratio of different concentration gradients of the two Bt proteins Bioassays were performed for 1:1, 1:2, 1:3, 2:1, 3:1.

Wherein, surface smearing method is used to conduct bioassays on cotton bollworm larvae with different resistance levels, and the inoculation amount of the cotton bollworm larvae is more than 24 cotton bollworm larvae/unit volume of medicine. The artificial feed for the cultivation of the cotton bollworm larvae is an improved cotton bollworm feed, and the feed composition includes corn flour, soybean powder, yeast powder, sucrose, and vitamins (for the specific formula, refer to formula 4 in "Improvement of Cotton Bollworm Technology Artificially Raising", plant Protection, Liang Gemei et al., 1999); the conditions for culturing cotton bollworm larvae are temperature 27±2°C, humidity 75±10%, light 14:10(L:D)h.

In step (4), use the DPS software to calculate the virulence regression equation to obtain the LC ₅₀ value.

In step (4), calculate the co-toxicity coefficient (CTC) according to the calculation method proposed by Sun and Johnson (1960) to express the synergistic effect:

According to the formula: CTC=(actual TI/theoretical TI)×100

TI (Toxicity Index) is the toxicity index.

Assuming that A is a calibrator, its TI=100; the TI value of B is: LC ₅₀ of A / LC ₅₀ of B × 100;

The actual TI of the mixture of two Bt proteins is LC ₅₀ of A / LC ₅₀ of the mixture × 100; the theoretical TI of the mixture of two Bt proteins = TI _A × A% in the mixture + TI _B × B% in the mixture.

Wherein, A% and B% are the mass percentages of the two Bt proteins A and B in the mixture, respectively.

Whether synergy is judged according to the following criteria: 80<CTC<120 means additive effect; CTC>120 means synergistic effect, the greater the number of CTCs, the greater the synergistic effect; CTC<80 means antagonistic effect.

Among them, for the sensitive strain cotton bollworm 96S, according to the method of the invention patent application with the application publication number CN102981001A, the optimal concentration range is calculated as: the concentration of Cry1Ac protein and Cry2Ab protein to control cotton bollworm is 1.75-3.65 μg/cm ² , then Set Cry2Ab and Cry1Ac protein different concentration gradient ratio to be 1:1, 1:2, 1:3, 2:1, 3:1 respectively, carry out bioassay; Utilize DPS software to calculate LC ₅₀ ; According to Sun and Johnson (1960 ) to calculate the CTC by the calculation method of expressing the synergistic effect proposed in 2010.

When the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=261.38>120 is synergistic; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, CTC=208.71>120 is synergistic; the concentration ratio of Cry1Ac and Cry2Ab When the concentration ratio of Cry1Ac to Cry2Ab is 2:1, CTC=170.90>120, it is synergistic effect; when the concentration ratio of Cry1Ac to Cry2Ab is 3:1, CTC =183.54>120, for synergistic effect. Therefore, for the sensitive strain of cotton bollworm 96S, both Bt protein concentrations Cry1Ac:Cry2Ab=1:1, 1:2, 2:1 and 3:1 have synergistic effects; but Cry1Ac:Cry2Ab=1:1 synergistic effect The most obvious.

Among them, for the resistant strain of cotton bollworm BtR1 strain Cry1Ac resistant cotton bollworm, according to the method of the invention patent application with the application publication number CN102981001A, the optimal concentration range is calculated as follows: Cry1Ac protein: 0.64-3.79 μg/cm ² , Cry2Ab protein :3.44-4.76μg/cm ² , then set the different concentration gradient ratios of Cry2Ab and Cry1Ac proteins to be 1:1, 1:2, 1:3, 2:1, and 3:1, respectively, for biological assay. Use DPS software to calculate LC ₅₀ ; calculate CTC according to the calculation method of expressing synergistic effect proposed by Sun and Johnson (1960).

When the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=129.65>120, it is a synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, 80<CTC=83.92<120, it is an additive effect; Cry1Ac and Cry2Ab When the concentration ratio is 1:3, 80<CTC=82.37<120 is an additive effect; when the concentration ratio of Cry1Ac and Cry2Ab is 2:1, CTC=108.68>120 is a synergistic effect; the concentration ratio of Cry1Ac and Cry2Ab is 3 : 1, CTC=141.05>120, which is synergistic effect. Therefore, for the resistant strain of cotton bollworm, the two drug concentrations of Cry1Ac:Cry2Ab=1:1, 2:1 and 3:1 all had synergistic effects, and the effect was the best when Cry1Ac:Cry2Ab=3:1.

The method for screening and controlling the optimal concentration ratio of two Bt proteins of cotton bollworm of the present invention has the following technical effects:

The method for screening the optimal concentration ratio of two Bt proteins for preventing and controlling cotton bollworms in the present invention is highly operable, accurate and efficient, and can clarify the concentration ratio of Bt proteins for cotton bollworms in different regions and with different resistance levels. It can be widely used in the selection of Bt protein or other protein ratios in trans-bivalent cotton to increase the insecticidal effect and delay resistance.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

If not specified, the experimental materials, reagents and instruments used in the examples of the present invention are all commercially available. If not specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 Optimal concentration ratio of two Bt proteins to sensitive cotton bollworm

1.1 Cotton bollworm for testing

The newly hatched larvae of the sensitive strain 96S of cotton bollworm.

1.2 Concentration ratio of Cry1Ac and Cry2Ab proteins

Calculate the optimum concentration range according to the method of the invention patent application with the application publication number CN102981001A. The concentration of Cry1Ac protein and Cry2Ab protein to control the sensitive strain 96S of cotton bollworm is 1.75-3.65μg/cm ² . For the two protein factors, according to the concentration gradient Cry1Ac:Cry2Ab=1:1, 1:2, 1:3, 2:1, and 3:1, bioassays were performed with 3 replicates.

1.3 Bioassay methods

Bioassays were performed using the surface smear method. The method is as follows: first add 1ml of unsolidified artificial feed into a 24-well plate (the artificial feed mainly consists of corn flour, soybean powder, yeast powder, sucrose, vitamins, etc., and the formula is referred to formula 4 in Liang Gemei et al. (1999) (Liang Gemei , Tan Weijia, Guo Yuyuan, 1999. Improvement of the artificial feeding technology of cotton bollworm. Plant Protection, 25(2):15–17)), after the feed solidified, then according to the concentration gradient Cry1Ac:Cry2Ab=1:1, 1:2, The protein mixture was added in 1:3, 2:1, and 3:1 ratios. The mixed protein was 60 μl/well, and the 24-well plate was shaken gently after adding, so that the mixed protein was evenly spread on the feed surface. Each treatment received 24 newly hatched larvae, set three repetitions, and checked the mortality of larvae on the 7th day. The conditions for culturing cotton bollworm larvae were temperature at 27°C, humidity at 75%, and light at 14:10(L:D)h.

1.4 Data Analysis

According to the mortality rate under different ratios, the LC ₅₀ was calculated by using DPS software.

According to the calculation method of expressing synergistic effect proposed by Sun and Johnson (1960), according to the formula: CTC=(actual TI/theoretical TI)×100

TI (Toxicity Index) is the toxicity index

Assuming that A is a calibrator, its TI=100; the TI value of B is: LC ₅₀ of A / LC ₅₀ of B × 100;

The actual TI of the mixture of the two agents is LC ₅₀ of A/LC ₅₀ of the mixture × 100; the theoretical TI of the two agents = TI _A × A% in the mixture + TI _B × B% in the mixture.

Wherein, A%, B% are respectively the mass percent of A, B in the mixture.

Calculate the CTC, and then judge whether it is synergistic according to the following criteria: 80<CTC<120, it is an additive effect; CTC>120, it is a synergistic effect, the larger the number of CTCs, the greater the synergistic effect; CTC<80, it is an antagonistic effect effect.

1.5 LC ₅₀ for the sensitive strain 96S of cotton bollworm

For the sensitive strain of cotton bollworm 96S, the LC ₅₀ of Cry1Ac = 0.93 μg/ml; the LC ₅₀ of Cry2Ab = 16.26 μg/ml was calculated by using DPS to calculate the mortality rate (Table 1);

1.6 Screening for synergistic effect

The Bt protein is Cry1Ac protein and Cry2Ab protein, when the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=261.38>120, which is a synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, CTC=208.71>120 , is the synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:3, CTC=69.40<80, it is antagonism; when the concentration ratio of Cry1Ac and Cry2Ab is 2:1, CTC=170.90>120, it is synergistic effect; When the concentration ratio with Cry2Ab is 3:1, CTC=183.54>120, which is a synergistic effect. Therefore, for the sensitive strain of cotton bollworm 96S, both Bt protein concentrations Cry1Ac:Cry2Ab=1:1, 1:2, 2:1 and 3:1 have synergistic effects; but Cry1Ac:Cry2Ab=1:1 synergistic effect The most obvious.

Table 1 LC ₅₀ of two Bt proteins mixed with different concentrations

Note: 95% CL is the 95% confidence limit.

Example 2 Optimum concentration ratio of two Bt proteins in resistant cotton bollworm

1.1 Cotton bollworm for testing

Newly hatched larvae of the resistant strain of cotton bollworm-BtR ₁ strain Cry1Ac resistant cotton bollworm larvae.

1.2 Concentration ratio of Cry1Ac and Cry2Ab proteins

According to the method of the invention patent application with the application publication number CN102981001A, the optimal concentration range is calculated, Cry1Ac protein: 0.64-3.79 μg/cm ² , Cry2Ab protein: 3.44-4.76 μg/cm ² , for the two protein factors, according to the concentration gradient Cry1Ac:Cry2Ab=1:1, 1:2, 1:3, 2:1, and 3:1, bioassays were performed with 3 replicates.

1.3 Bioassay methods

Bioassays were performed using the surface smear method. The method is as follows: first add 1ml of unsolidified artificial feed into a 24-well plate (the artificial feed mainly consists of corn flour, soybean flour, yeast powder, sucrose, vitamins, etc., and the formula refers to formula 4 in Liang Gemei et al. (1999)), After the feed is coagulated, add the mixed protein according to the ratio of concentration gradient Cry1Ac:Cry2Ab=1:1, 1:2, 1:3, 2:1, and 3:1. The mixed protein was 60 μl/well, and the 24-well plate was shaken gently after adding, so that the mixed protein drug solution was evenly spread on the feed surface. Each treatment received 24 newly hatched larvae, set three repetitions, and checked the mortality of larvae on the 7th day. The conditions for culturing cotton bollworm larvae were temperature at 27°C, humidity at 75%, and light at 14:10(L:D)h.

1.4 Data Analysis

According to the mortality rate under different proportions, use DPS software to calculate LC ₅₀ , and calculate CTC according to the calculation method of expressing synergistic effect proposed by Sun and Johnson (1960), and then judge whether synergistic effect is judged according to the following criteria: 80<CTC<120 means additive effect; CTC>120 means synergistic effect, the greater the number of CTCs, greater synergistic effect; CTC<80 means antagonistic effect.

1.5 LC ₅₀ for resistant strains of cotton bollworm

For the resistant BtR ₁ strain of cotton bollworm, the LC ₅₀ of Cry1Ac = 2045.84 μg/ml and the LC ₅₀ of Cry2Ab = 79.06 μg/ml were obtained by calculating the mortality rate and using DPS (Table 1).

1.6 Screening for synergistic effect

The Bt protein is Cry1Ac protein and Cry2Ab protein, when the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=129.65>120, which is a synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, 80<CTC=83.92 <120, it is an additive effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:3, 80<CTC=82.37<120, it is an additive effect; when the concentration ratio of Cry1Ac and Cry2Ab is 2:1, CTC=108.68>120, it is Synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 3:1, CTC=141.05>120, which is synergistic effect. Therefore, for the resistant strain of cotton bollworm, the two drug concentrations of Cry1Ac:Cry2Ab=1:1, 2:1 and 3:1 all had synergistic effects, and the effect was the best when Cry1Ac:Cry2Ab=3:1.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. the screening method of two kinds of Bt protein optimal concentration ratios of preventing and treating cotton bollworm, it is characterized in that, described method comprises the following steps: (1) Select two Bt proteins for the control of cotton bollworm; (2) Inoculate cotton bollworm larvae with different resistance levels, and calculate the optimal concentration range of the mixed use of the two Bt proteins when controlling cotton bollworm larvae with different resistance levels; (3) According to the optimal concentration range, different concentration gradient ratios of the two Bt proteins were set, and cotton bollworm larvae with different resistance levels were inoculated for bioassay; (4) Observe the death of larvae, calculate the mortality rate, LC ₅₀ and co-toxicity coefficient CTC, and determine the control effect of different concentration ratios of the two Bt proteins. 2 . The method according to claim 1 , wherein in step (1), the Bt protein is any two of Cry2Ab protein, Cry1Ab protein, Cry1F protein, Vip3A protein and Cry1Ac protein. 3. The method according to claim 2, characterized in that, in step (1), the Bt protein is Cry2Ab protein and Cry1Ac protein. 4. The method according to claim 1, characterized in that, in step (2), the method for calculating the optimum concentration range for mixing the two Bt proteins comprises the following steps: a, select two kinds of Bt protein for preventing and treating cotton bollworm; b. Inoculation of cotton bollworm larvae with different resistance levels; c, observe the larva death situation, calculate the mortality rate; d. Using the two-factor D-saturation method to establish a regression equation, evaluate the control effects of the two Bt proteins, and obtain the optimal concentration range for the mixed use of the two Bt proteins. 5. The method according to claim 1, characterized in that, in steps (2) and (3), the cotton bollworm larvae with different resistance levels are newly hatched larvae of sensitive strains of cotton bollworm or newly hatched larvae of resistant strains of cotton bollworm . 6. The method according to claim 1, characterized in that, in step (3), within the optimal concentration range, the ratio of different concentration gradients of the two Bt proteins is set to 1:1, 1:2, 1 :3, 2:1, 3:1 for bioassay. 7. The method according to claim 1, characterized in that, in step (3), bioassays are carried out on cotton bollworm larvae with different resistance levels by surface smearing method, and the inoculum size of the cotton bollworm larvae is 24 The above cotton bollworm larvae/unit volume of drug; the conditions for culturing the cotton bollworm larvae are temperature 27±2°C, humidity 75±10%, and light 14:10h. 8. The method according to claim 1, characterized in that in step (4), it is judged whether synergism is based on the following criteria: 80<CTC<120, it is an additive effect; CTC>120, it is a synergistic effect, CTC The larger the number, the greater the synergistic effect; CTC<80, it is an antagonistic effect. 9. according to the method described in any one of claim 1-8, it is characterized in that, for sensitive strain cotton bollworm, when described two kinds of Bt albumen are Cry1Ac protein and Cry2Ab albumen, Cry1Ac albumen and Cry2Ab albumen control the most of cotton bollworm The best concentration is 1.75-3.65μg/cm ² ; When the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=261.38>120, it is a synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, CTC=208.71>120, it is a synergistic effect; When the concentration ratio of Cry1Ac to Cry2Ab is 2:1, CTC=170.90>120, it is synergistic effect; when the concentration ratio of Cry1Ac to Cry2Ab is 3:1, CTC= 183.54>120, for the synergistic effect. 10. according to the described method of claim 1-8 any one, it is characterized in that, for BtR ₁ strain Cry1Ac resistance cotton bollworm, when described two kinds of Bt proteins are Cry1Ac protein and Cry2Ab protein, Cry1Ac protein controls the effect of cotton bollworm The optimal concentration is 0.64-3.79 μg/cm ² , and the optimal concentration of Cry2Ab protein for controlling cotton bollworm is 3.44-4.76 μg/cm ² ; When the concentration ratio of Cry1Ac and Cry2Ab is 1:1, CTC=129.65>120, it is a synergistic effect; when the concentration ratio of Cry1Ac and Cry2Ab is 1:2, 80<CTC=83.92<120, it is an additive effect; the concentration of Cry1Ac and Cry2Ab When the ratio is 1:3, 80<CTC=82.37<120 is an additive effect; when the concentration ratio of Cry1Ac and Cry2Ab is 2:1, CTC=108.68>120 is a synergistic effect; the concentration ratio of Cry1Ac and Cry2Ab is 3: When 1, CTC=141.05>120, it is a synergistic effect.